Trans-kingdom transposition of the maize dissociation element

Transposons are very valuable tools for genetic manipulation. However, the number of transposable elements that have been suitably adapted for experimental use is insufficient and the spectrum of heterologous hosts in which they have been deployed is restricted. To date, only transposons from animal hosts have been utilized in heterologous animal species and transposons of plant origin have been used in plant genetics. There has been no experimental evidence that any of the known elements could transpose in hosts belonging to both kingdoms. Here we demonstrate that the maize Dissociation (Ds) element is capable of effective Activator (Ac) transposase-mediated transposition in the zebrafish Danio rerio, yielding remarkable germline transmission rates. In addition, mammalian cells were also found to be conducive to Ds transposition. Furthermore, we demonstrate that nuclear localization of Ac transposase is essential for genomic Ds transposition. Our results support the hypothesis that Ac/Ds elements do not rely on host-specific factors for transposition and that host factors involved in their mobility mechanism are widely conserved. Finally, even in vertebrate cells, the Ac/Ds system displays accurate transposition, large-fragment carrying capacity, high transposition frequencies, efficient germline transmission, and reporter gene expression, all of which are advantageous for various genetic applications and animal biotechnology.     

Transposition of the maize activator element in transgenic rice plants.

Transposition of the maize Activator (Ac) element was observed in transgenic rice. After protoplast transformation, Ac excision from an interrupted hygromycin phosphotransferase gene was monitored by appearance of the hygromycin-resistant colonies. The frequency of Ac excision, based on the biological assay was up to 19%. Southern hybridization analysis indicated that at least one copy per genome of the hygromycin-resistance gene was reconstituted after Ac excision and that the transposed Ac element was reintegrated into the rice genome. Analysis of DNA sequences at 14 empty donor sites indicated that the Ac element was excised in rice in a similar manner as maize. The excision of an Ac mutant in which a 1.3 kbp Tn903 fragment was inserted at a unique BamHI site so as to disrupt binding of the putative transposase was not detected by DNA analysis. These results demonstrated that the maize Ac element might be used as an effective heterologous transposon for mutagenesis and gene tagging in rice, an important food crops.     

Phenotypic assay for excision of the maize controlling element Ac in tobacco

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 µg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed ˜25% as many calli resistant to 100 or 200 µg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac did not form any calli resistant to 200 µg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.     

Repair of P element ends following hybrid element excision leads to recombination in Drosophila melanogaster

P elements are thought to replicate themselves starting with the association of the left and right ends, followed by a cut-copy-paste process. An abnormal form of this process has been shown to occur when the associated left and right ends come from sister elements rather than from the same element, leading to formation of a 'hybrid element.' These ends can insert nearby in the genome to produce recombination, with associated structural changes. We have previously increased the frequency of such 'hybrid element insertion' by combining end-deleted elements in trans in a genotype with a left-end on one chromosome and a right-end on the homologous chromosome. Although many recombinants produced by this genotype have structural changes expected with insertion, nearly 50% of the predicted insertional recombinants contain no structural change. We present evidence using RFLP markers closely linked to the end-deleted elements that in these cases the P element ends dissociate before insertion, and are subsequently ligated together following a process analogous to synthesis-dependent strand annealing. The results suggest that broken ends containing P elements are resolved by the same repair process as ends not containing P elements, and that such repair from hybrid element events may occur in the majority of cases.     

A hyperactive transposase of the maize transposable element activator (Ac)

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.     

Factors affecting the excision frequency of the maize transposable element Ds in Arabidopsis thaliana

A two-element transposon system based on the maize elements Ac and Ds is currently being used for insertional mutagenesis in Arabidopsis. With the aim of making this system as efficient as possible we have continued to analyse several parameters which affect Ds activity in Arabidopsis. The influence of genomic position on Ds excision has been analysed in five lines carrying Ds integrated in different genomic locations. Differences in both somatic and germinal excision were observed between the different lines. The relationship between somatic and germinal excision, the timing of excision events and environmental influences on transposition frequency have been investigated. The effect of varying dosage of the different elements was also analysed. A strong positive dosage effect was observed for the transposase source, but not for the Ds element. Analysis of germinal excision events showed that the majority of them occurred very late in the development of the plant, resulting in the majority of Ds transpositions being independent events.     

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy ( wx ) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac -containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis , including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds -containing plants will be useful tools in dissecting plant DNA repair processes.     

Factors affecting the excision frequency of the maize transposable element Ds in Arabidopsis thaliana

A two-element transposon system based on the maize elements Ac and Ds is currently being used for insertional mutagenesis in Arabidopsis. With the aim of making this system as efficient as possible we have continued to analyse several parameters which affect Ds activity in Arabidopsis. The influence of genomic position on Ds excision has been analysed in five lines carrying Ds integrated in different genomic locations. Differences in both somatic and germinal excision were observed between the different lines. The relationship between somatic and germinal excision, the timing of excision events and environmental influences on transposition frequency have been investigated. The effect of varying dosage of the different elements was also analysed. A strong positive dosage effect was observed for the transposase source, but not for the Ds element. Analysis of germinal excision events showed that the majority of them occurred very late in the development of the plant, resulting in the majority of Ds transpositions being independent events.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair

CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.     

Developmental and genetic aspects of Mutator excision in maize

The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.     

Aerenchyma formation in maize roots

Maize (Zea mays L.) is generally considered to be a plant with aerenchyma formation inducible by environmental conditions. In our study, young maize plants, cultivated in various ways in order to minimise the stressing effect of hypoxia, flooding, mechanical impedance or nutrient starvation, were examined for the presence of aerenchyma in their primary roots. The area of aerenchyma in the root cortex was correlated with the root length. Although 12 different maize accessions were used, no plants without aerenchyma were acquired until an ethylene synthesis inhibitor was employed. Using an ACC-synthase inhibitor, it was confirmed that the aerenchyma formation is ethylene-regulated and dependent on irradiance. The presence of TUNEL-positive nuclei and ultrastructural changes in cortical cells suggest a connection between ethylene-dependent aerenchyma formation and programmed cell death. Position of cells with TUNEL-positive nuclei in relation to aerenchyma-channels was described.     

Reactivation of a silenced minimal Mutator transposable element system following low-energy nitrogen ion implantation in maize

In maize, Mutator transposable elements are either active or silenced within the genome. In response to environmental stress, silenced Mutator elements could be reactivated, leading to changes in genome structure and gene function. However, there is no direct experimental evidence linking environmental stress and Mutator transposon reactivation. Using a maize line that contains a single inactive MuDR and a lone nonautonomous Mutator element, a Mu1 insertion in the recessive reporter allele a1-mum2 in an inactive Mutator background, we directly assessed Mutator reactivation following low-energy nitrogen ion implantation. We observed that N⁺ implantation decreased cytosine methylation in MuDR terminal inverted repeats and increased expression of mudrA and mudrB. Both changes were associated with increased transpositional activity of MuDR through reactivation of the inactive minimal Mutator transposable element system. This study provides direct evidence linking environmental stress agents and Mutator transposon mobilization in maize. In addition, the observed changes to DNA methylation suggest a new mechanism for mutations by low-energy ion implantation.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

Nitrate reductase activity in chicory roots following excision

In young chicory plantlets (Cichorium intybus L. Witloof cv.Flash), nitrate assimilation takes place mainly in the roots.Nitrate reductase activity (NRA) was measured in roots deprivedof shoot control by excision and transferred into a sucrose-containingmedium. Such a treatment resulted in a drop of about 60% ofNRA within 3 h. The level of NR protein decreased after 12 hand the level of NR-mRNA after several days. This adaptationof nitrate assimilation to excision was affected by a phosphorylation-dephosphorylationmechanism as shown by increased sensitivity to magnesium ofin vitro NRA. Okadaic acid, a serinethreonine protein phosphatasesinhibitor, enhanced the decrease of NRA. Conversely, staurosporine,a serine-threonine protein kinases inhibitor, antagonized theinhibition of NRA. This suggests that excision caused a rapidinactivation of NRA in roots of chicory by modifying the phosphorylationbalance towards a phosphorylated NR form which could enter aninactive complex. Key words: Chicory, nitrate reductase, staurosporine