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.     

Gametophyte-specific transposition of the maize Ds element in transgenic tobacco

To assess the potential advantages of a transposon-tagging system based on gametophyte-specific transposition a fusion between the anther-specific Arabidopsis thaliana apg promoter and the maize Ac transposase gene was constructed and introduced into tobacco. The ability of this transposase source to activate Ds transposition in a developmentally controlled manner was monitored by crossing to plants harbouring the cell autonomous excision marker gene construct, Ds —SPT. A number of fully green, streptomycin-resistant seedlings resulting from germinal transposition events were observed in the progeny of apg -TPase x Ds —SPT F1 plants. Streptomycin-resistant sectors were not observed in either F₁ seedlings or F₂ progeny, indicating a complete lack of somatic excision. Further crosses of apg —TPase sources to plants containing Ds—bar herbicide selection excision marker constructs gave reproducible gametophytic excision frequencies of up to 0.3%. Sequencing of Ds excision sites from F₂ seedlings derived from single F₁ plants revealed various sequence alterations in the original Ds insertion 'footprint' indicative of independent Ds excision events. Independent re-insertion was confirmed by Southern analysis of F₂ siblings. It is concluded that apg -controlled Ac transposase expression activates male gametophyte-specific Ds transposition.     

The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines.

Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac). Here, we describe experiments designed to determine the basis for the high activity of Tag1. The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity. Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed. The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines. This result was maintained through at least six generations after transformation. These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active. Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1. Tag1 elements were found in only a few A. thaliana ecotypes but were present in four other Arabidopsis species.     

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.     

Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue

The development of a barley ( Hordeurn vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation ( Ac/Ds ) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co-transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uid A reporter gene ( Gus ), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.     

Ac transposition is impaired by a small terminal deletion

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.     

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.     

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.