The maize Activator/Dissociation system is functional in hexaploid wheat through successive generations

The aim of the present study was to provide useful background information and evidence of the functionality of the maize Activator/Dissociation (Ac/Ds) system in hexaploid wheat. Two transgenic parental wheat lines, one harbouring the immobilised Ac element (iAc) and the other the Ds element (pUbi[Ds-uidA]bar), were crossed. Transient GUS assays confirmed that the iAc transposase is active in hexaploid wheat. Selected F1 and F2 lines were analysed by PCR using primers specific to Ac, uidA and bar genes. The primer pair Ubi/bar-tag was used to detect excision of the Ds-uidA sequence, which occurred at a frequency of 39% in the F1 generation. Lines free of Ac and showing evidence of Ds excision were subject to Southern analysis, which indicated that at least one transposition event might have occurred in these lines. Although more evidence is required to unequivocally support the reintegration of the Ds element in the wheat genome, the evidence presented here nevertheless demonstrates the effectiveness and potential value of using this system to tag genes in wheat.

Regulation of Activator/Dissociation Transposition by Replication and DNA Methylation

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids (“chromatid selectivity”). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.

Analysis of Extrachromosomal Ac/Ds Transposable Elements

The mechanism of transposition of the maize Ac/Ds elements is not well understood. The true transposition intermediates are not known and it has not been possible to distinguish between excision models involving 8-bp staggered cuts or 1-bp staggered cuts followed by hairpin formation. In this work, we have analyzed extrachromosomal excision products to gain insight into the excision mechanism. Plasmid rescue was used to demonstrate that Ds excision is associated with the formation of circular molecules. In addition, we present evidence for the formation of linear extrachromosomal species during Ds excision. Sequences found at the termini of circular and linear elements showed a broad range of nucleotide additions or deletions, suggesting that these species are not true intermediates. Additional nucleotides adjacent to the termini in extrachromosomal elements were compared to the sequence of the original donor site. This analysis showed that: (1) the first nucleotide adjacent to the transposon end was significantly more similar to the first nucleotide flanking the element in the donor site than to a random sequence and (2) the second and farther nucleotides did not resemble the donor site. The implications of these findings for excision models are discussed.

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.Key words: transposon, Ds excision, Ac transposase transcript level, transgenic callus, wheat.

Excision Patterns of Activator (Ac) and Dissociation (Ds) Elements in Zea mays L.: Implications for the Regulation of Transposition

The pattern of aleurone variegation of maize kernels carrying Ac and bz-m2(DI) as reporter allele for Ac activity depends on the dosage of both Ac and Ds. Alterations of Ac dosage can abolish Ds excision at certain times and allow it to occur at other times. wx-m7 and wx-m9 are different Ac insertions in the Waxy gene which have different dosage effects on Ds excision. Kernels, heterozygous for the two Ac alleles and being either wx-m7/wx-m7/wx-m9 or wx-m9/wx-m9/wx-m7 exhibit characteristic patterns of predominantly late excisions; this is in strong contrast to the pattern of early excisions present on wx-m7/wx-m7/wx-m7 homozygotes. This observation supports the hypothesis that the Ac alleles express different amounts of transposase (TPase) during development and that above a certain level of TPase transposition is inhibited. Furthermore, experimental results suggest that the frequency of Ac-induced events is influenced by the dosage and composition of the transactivated Ds or Ac allele. Thus, transposition frequency seems not to be exclusively determined in trans by the amount of active TPase, but also by specific cis-acting properties of the TPase substrate.