Transposase binding site methylation in the epigenetically inactivated <i>Ac</i> derivative <i>Ds‐cy</i>

Wang, Lihua; Kunze, Reinhard

doi:10.1046/j.1365-313x.1998.00060.x

Cited by 15 publications

(5 citation statements)

References 29 publications

(50 reference statements)

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“…In addition, treatment with methylation inhibitors (5-azacytidine or 5-azacytidine þ ethionine) did not affect Tam3 excision frequency in Antirrhinum callus (Hashida et al, 2005). In the case of Ac/Ds, a hemimethylated state for the TPase binding sites promotes transposition, and even completely methylated Ds is able to transpose after replication (Wang and Kunze, 1998;Ros and Kunze, 2001). These results with Ac/Ds suggest that the arrest of Tam3 transposition at high temperature is not likely to be the direct result of methylation, because high temperature accelerates cell division and replication, which promotes (at least transient) reductions in methylation.…”

Section: Role Of Methylation In the Ltdt Of Tam3mentioning

confidence: 99%

The Temperature-Dependent Change in Methylation of theAntirrhinumTransposon Tam3 Is Controlled by the Activity of Its Transposase

et al. 2005

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The Antirrhinum majus transposon Tam3 undergoes low temperature-dependent transposition (LTDT). Growth at 158C permits transposition, whereas growth at 258C strongly suppresses it. The degree of Tam3 DNA methylation is altered somatically and positively correlated with growth temperature, an exceptional epigenetic system in plants. Using a Tam3-inactive line, we show that methylation change depends on Tam3 activity. Random binding site selection analysis and electrophoretic mobility shift assays revealed that the Tam3 transposase (TPase) binds to the major repeat in the subterminal regions of Tam3, the site showing the biggest temperature-dependent change in methylation state. Methylcytosines in the motif impair the binding ability of the TPase. Proteins in a nuclear extract from plants grown at 158C but not 258C bind to this motif in Tam3. The decrease in Tam3 DNA methylation at low temperature also requires cell division. Thus, TPase binding to Tam3 occurs only during growth at low temperature and immediately after DNA replication, resulting in a Tam3-specific decrease in methylation of transposon DNA. Consequently, the Tam3 methylation level in LTDT is regulated by Tam3 activity, which is dependent on the ability of its TPase to bind DNA and affected by growth temperature. Thus, the methylation/demethylation of Tam3 is the consequence, not the cause, of LTDT.

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Section: Role Of Methylation In the Ltdt Of Tam3mentioning

confidence: 99%

The Temperature-Dependent Change in Methylation of theAntirrhinumTransposon Tam3 Is Controlled by the Activity of Its Transposase

et al. 2005

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“…This chromatid selectivity can be explained by the methylation status of the transposon: a holomethylated transposon is unable to transpose, whereas the two daughter transposons produced by replication will be hemimethylated on distinct DNA strands and one of these hemimethylated elements is a preferred transposase substrate (Wang et al 1996;Wang and Kunze 1998;Ros and Kunze 2001). These ®ndings are consistent with our proposal that transposase acts upon one of the newly replicated transposon ends, while the other transposon end is unreplicated and not yet competent for transposition.…”

Section: A Model For Ac Transposition During Element Replicationmentioning

confidence: 99%

Ac transposition is impaired by a small terminal deletion

Xiao

Peterson

2002

Mol Gen Genomics

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In maize, the P1-vv allele specifies variegated pericarp and cob pigmentation, and contains an Ac transposable element inserted in the second intron of the P1-rr gene. Starting from P1-vv, we recovered a new allele, called P1-vv5145, which gives an extremely light variegated pericarp and cob phenotype. The P1-vv5145 allele contains an Ac element ( Ac5145) at the same position and in the same orientation as in the progenitor P1-vv allele; however, the P1-vv5145 allele has a 2-bp deletion which removes the last nucleotide (A) from the 3' end of the Ac element, and an adjacent flanking nucleotide (C) from the p1 intron. In crosses with a Ds tester stock, P1-vv5145 shows a normal ability to induce Ds transposition; however, Ac excision from P1-vv5145 is 3800-fold less frequent than from the progenitor P1-vv allele. Our results demonstrate that the alteration of the 3' terminal base strongly impairs Ac transposition. The P1-vv5145 allele thus provides a relatively stable source of Ac transposase for controlling Ds transposition in genetic experiments. In addition, we describe two further alleles ( P1-ww7B8, P1-ww9A146-3) that contain deletions of Ac and flanking p1 gene sequences. These latter deletions are larger and involve the 5' end of the the Ac element. A model is proposed to explain the formation of one-sided deletions as a consequence of Ac transposition during replication of the element.

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“…The varying transposition frequencies result from the complex regulation of Ac activity, including epigenetic inactivation by the host plant. The DNA methylation state affects the Ac promoter activity (Kunze et al 1988) and the binding of the AcTPase to the subterminal binding sites (Wang et al 1996;Wang and Kunze 1998;Ros and Kunze 2001). Moreover, there is no linear relationship between AcTPase levels and transposition frequency.…”

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confidence: 99%

A Hyperactive Transposase of the Maize Transposable ElementActivator(Ac)

Lazarow

Weimer

et al. 2012

Genetics

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Activator/Dissociation (Ac/Ds) transposable elements from maize are widely used as insertional mutagenesis and gene isolation tools in plants and more recently also in medaka and zebrafish. They are particularly valuable for plant species that are transformation-recalcitrant and have long generation cycles or large genomes with low gene densities. Ac/Ds transposition frequencies vary widely, however, and in some species they are too low for large-scale mutagenesis. We discovered a hyperactive Ac transposase derivative, AcTPase 4x , that catalyzes in the yeast Saccharomyces cerevisiae 100-fold more frequent Ds excisions than the wild-type transposase, whereas the reintegration frequency of excised Ds elements is unchanged (57%). Comparable to the wild-type transposase in plants, AcTPase 4x catalyzes Ds insertion preferentially into coding regions and to genetically linked sites, but the mutant protein apparently has lost the weak bias of the wild-type protein for insertion sites with elevated guanine-cytosine content and nonrandom protein-DNA twist. AcTPase 4x exhibits hyperactivity also in Arabidopsis thaliana where it effects a more than sixfold increase in Ds excision relative to wild-type AcTPase and thus may be useful to facilitate Ac/Ds-based insertion mutagenesis approaches. DNA transposons are widely used in plants and animals as functional genomics tools and gene transfer vehicles. In plants, transposable elements are particularly valuable when large-scale T-DNA insertion mutagenesis is not feasible, e.g., for transformation-recalcitrant species or plants with long generation cycles. The success of transposon insertion mutagenesis strategies depends on high forward mutagenesis rates and a favorable distribution of novel insertions. As forward mutagenesis rates are frequently limited by transposase activity, attempts were made to find hyperactive transposase mutants. For some transposons, such mutants were fortuitously found; in other instances, systematic screening approaches and molecular evolution were successful (Goryshin and Reznikoff 1998;Beall et al. 2002;Baus et al. 2005;Keravala et al. 2006;Mates et al. 2009).The maize Activator/Dissociation (Ac/Ds) transposable elements have been widely used in plants for gene tagging and functional genomics approaches because they are active in numerous plant species, integrate preferentially into or near to coding regions, and frequently transpose to genetically linked sites, enabling local saturation mutagenesis approaches (reviewed in Kunze and Weil 2002). The successful introduction of Ac/Ds elements into yeast revealed that application of these elements is not restricted to plants (Weil and Kunze 2000). Their recent adoption as tools for transgenesis and the generation of gene trap lines in the teleost fishes zebrafish and medaka further emphasized the wide range functionality of Ac/Ds elements (Emelyanov et al. 2006;Boon Ng and Gong 2011;Froschauer et al. 2012).An impediment for a universal application is the variability of Ac/Ds transposition fr...

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Transposase binding site methylation in the epigenetically inactivated Ac derivative Ds‐cy

Cited by 15 publications

References 29 publications

The Temperature-Dependent Change in Methylation of theAntirrhinumTransposon Tam3 Is Controlled by the Activity of Its Transposase

The Temperature-Dependent Change in Methylation of theAntirrhinumTransposon Tam3 Is Controlled by the Activity of Its Transposase

Ac transposition is impaired by a small terminal deletion

A Hyperactive Transposase of the Maize Transposable ElementActivator(Ac)

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